Involvement Golgi Apparatus Synthesis Secretion of ... · Plant Physiol. (1975) 55, 536-541 Involvement ofthe Golgi Apparatus in the Synthesis andSecretion ofHydroxyproline-rich Cell

Plant Physiol. (1975) 55, 536-541

Involvement of the Golgi Apparatus in the Synthesisand Secretion of Hydroxyproline-rich CellWall Glycoproteins1 2 3

Received for publication June 26, 1974 and in revised form October 17, 1974

MICHAEL GARDINER4 AND MAARTEN J. CHRISPEELSDepartment of Biology, John Muir College, University of California, San Diego, La Jolla, California 92037

ABSTRACT

Pulse labeling of carrot root phloem parenchyma (Daucuscarota L. cv. Nantes) tissue with '4C-proline followed by frac-tionation of the cytoplasmic organelles on sucrose gradientswas used to determine the identity of the membranous organ-elies involved in the secretion of the hydroxyproline-rich gly.coproteins of the cell wall. Identification of the organelles wasdone through electron-microscopical observations and throughthe localization of marker enzymes on the sucrose gradients.Enrichment of the organelles involved in secretion was de-termined by measuring the percentage of the incorporatedradioactivity present as '4C-hydroxyproline. The Golgi appa-ratus (dictyosome) was found to be a major site of glycopro-tein transport. This identification was based on the observedenrichment of dictyosomes paralleling the purification ofnewly synthesized cell-wall glycoproteins. A marker enzymefor the Golgi apparatus, inosinediphosphatase, banded withthe newly synthesized cell wall glycoproteins on sequentialisopycnic and rate zonal sucrose gradients. Marker enzymesfor the endoplasmic reticulum and the plasma membrane wereclearly separated from the dictyosome-rich fraction. UDP-arabinose arabinosyl transferase, an enzyme involved in theglycosylation of the peptide moiety of this glycoprotein, alsobanded with the dictyosomes on both kinds of gradients. Theresults suggest an important role of the Golgi apparatus in thebiosynthesis and the secretion of the cell wall glycoproteins ofhigher plants.

The primary walls of plant cells contain a structural hy-droxyproline-rich glycoprotein called "extensin" (for reviewsee Lamport [12]). This protein is synthesized in the cytoplasmas a proline-rich polypeptide and the proline residues are hy-droxylated by a soluble peptidyl proline hydroxylase (20). The

'This investigation was supported by the Atomic Energy Com-mission under Contract AT(04-3)-34 PA 159.'A preliminary account of this work was presented at the annual

meeting of the American Association of Plant Physiologists in July1973 (8).

'This is paper No. 8 in a series on the "Synthesis and Secretionof Hydroxyproline-containing Proteins in Carrot." No. 7 appears asreference 4.

'Present address: Department of Biology, University of PugetSound, Tacoma, Wash. 98416.

hydroxyproline-residues formed in this way are subsequentlyglycosylated by a particulate UDP-arabinose arabinosyl trans-ferase (1 1). Each hydroxyproline residue acquires a short side-chain consisting of several arabinose residues (13). Secretionof the finished glycoprotein to the cell wall involves its transientassociation with one or more cytoplasmic organelles (3, 6). Theorganelles involved in the glycosylation of the polypeptide andin the secretion of the glycoprotein have not been identified.Dashek (6) examined this problem several years ago but hisresults were inconclusive. Secretion seemed to be mediated bysmooth membrane elements but he was unable to establishwhether these were derived from the endoplasmic reticulum,the plasma membrane, or the Golgi apparatus. Progress in themethodology of cell fractionation and the availability of newmarker enzymes prompted us to reinvestigate this question.Our results suggest that the Golgi apparatus plays a major rolein the biosynthesis and secretion of the hydroxyproline-richcell wall glycoproteins.

MATERIALS AND METHODS

Materials. Carrot roots (Daucus carota L. cv. Nantes) werepurchased in local supermarkets and stored at 4 C. Radioactivechemicals were obtained from New England Nuclear Co.:UDP-arabinose [L-arabinose-'4C(U)] (183 mCi/mmole); UDP-glucose [D-glucose-'4C(U)] (227 mCi/mmole); GDP-mannose[D-mannose-14C(U)] (167 mCi/mmole); UDP-galactose [D-galactose-'4C(U)] (254 mCi/mmole); and L-proline-"C(U) (233mCi/mmole) or ICN: GDP-glucose [D-glucose-"C(U)] (203mCi/mmole) and ICN isotope. Inosine diphosphate, NADH,and Cyt c were obtained from Sigma. Proteins precipitated withtrichloroacetic acid were collected on cellulose nitrate filtersobtained from Schleicher and Shuell (Type B-4).

Fractionation of Cytoplasmic Organelies. Carrot rootphloem parenchyma disks were prepared and incubated for24 hr at 30 C as described (3). The isolation and purificationof the membranous organelles was based on a modification ofthe methods of Ray et al. (18). Glutaraldehyde was includedin the homogenization medium to insure structural preserva-tion of the Golgi apparatus (15). The homogenization mediumconsisted of ice-cold 50 mm tris-Cl buffer (pH 8.0) containing10 mm KCI, 0.1 mm MgCI,, 1 mm Na2EDTA, 1 mm dithio-threitol, 1% dextran (mol wt 250,000), 0.1% BSA, 10% su-crose, and 0.5% glutaraldehyde. Glutaraldehyde was omittedin some experiments to determine its effect on the sedimenta-tion behavior of the cytoplasmic organelles and the activity ofmarker enzymes. The tissue was usually minced with a hand-held razor blade and then thoroughly chopped with a pair ofstainless steel razor blades mounted in an electrically driven

536

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THE GOLGI APPARATUS IN GLYCOPROTEINS

mechanical chopper. This operation was performed in a plasticPetri dish held on ice. After homogenization the brei wasfiltered through one layer of Miracloth to remove debris andwas centrifuged in the cold at 1OOOg for 5 min. The lOOOgsupernatant was used to further fractionate the cytoplasmicorganelles. Linear sucrose gradients were made by dissolvingsucrose (Merck) in the homogenization medium from whichglutaraldehyde had been omitted. Cytoplasmic organelleslayered on linear 20 to 50% (w/v) gradients were centrifugedto equilibrium by centrifugation at 27,000 rpm for 2 hr at 3 C(Spinco Rotor SW 27). For rate zonal separations the organelleswere layered on linear 20 to 35% (w/v) gradients and cen-trifuged for 20 min at 15,000 rpm (Spinco rotor SW 27.1).When necessary, organelles were concentrated onto a 50%sucrose cushion by centrifugation at 20,000 rpm for 20 min.In Vivo Labeling of Newly Synthesized Protein and Deter-

mination of Radioactivity in Protein-bound Proline and Hy-droxyproline. The tissue was thorouhgly rinsed and then incu-bated with "4C-proline (1 juCi/g of tissue) for 20 min at 30 Con a shaking waterbath. Chloramphenicol (50 ,ug/ml) wasroutinely included and does not affect the synthesis of cell wallproteins (3). Aliquots of the tissue homogenate or of gradientfractions were precipitated with an equal volume of 15% tri-chloroacetic acid and the precipitated proteins were collectedon cellulose nitrate filters. The filters were washed with 5%trichloroacetic acid, dried, and the radioactivity was deter-mined with a liquid scintillation counter after immersion ofthe dry filters in vials containing a toluene-based scintillationcocktail. The filters were then removed from the vials, washedwith toluene, dried, and hydrolyzed in sealed glass ampulescontaining 6 N HCI (autoclaved for 90 min at 120 C and 22psi). The HCI was removed by evaporation, and the prolineand hydroxyproline in the residue were separated by paperchromatography. Radioactivity in each amino acid was deter-mined and the percentage of radioactivity in proline and hy-droxyproline was calculated (3).Enzyme Assays. Gradient fractions were collected and as-

sayed for absorbancy at 280 nm (A.), for inosine diphos-phatase, a marker enzyme for the Golgi apparatus (7, 9), forNADH-Cyt c reductase, a marker enzyme for the endoplasmicreticulum (5), UDP-glucose and GDP-glucose glucosyl trans-ferases, marker enzymes for the plasma-membrane (23), andfor UDP-arabinose arabinosyl transferase, an enzyme involvedin the glycosylation of the cell wall glycoprotein (11).

IDPase activity was assayed by measuring phosphatase ac-tivity for 60 min at 35 C using 5 mM inosine-diP in 50 mMtris-Cl, pH 7.2, containing 100 mm KCI and 5 mm MgCI2.Phosphate released was determined by the method of Tausskyand Shorr (22) after protein precipitation with an equal volumeof 15% trichloroacetic acid.

Cyt c reductase activity was measured spectrophotometri-cally at room temperature by recording the rate of reduction ofoxidized Cyt c at 550 nm. The reaction mixture contained 50mm potassium phosphate, pH 7.5, 0.05 mm Cyt c, 0.5 mMNADH, and 1.6 mm KCN.

UDP-arabinose arabinosyl transferase activity was measuredby determining the radioactivity incorporated into trichloro-acetic acid-insolub'e products retained on cellulose nitratemembrane filters. The reaction was performed by adding 0.04juCi UDP-arabinose [L-arabinose-QC(U)] (183 mCi/mmole)to 250 ,lI of membrane fraction (0.86 ,um final UDP-arabinoseconcentration) and incubating 15 min at 35 C. The reactionwas stopped by adding an equal volume of 15% trichloroaceticacid, precipitating 20 min in an ice bath, and collecting theresidue on a membrane filter. UDP-glucose and GDP-glucoseglucosyl transferase activity was determined similarly but the

reaction was performed in 100 mm tris-Cl, pH 8, 1 mMNa2EDTA, 1 mm dithiothreitol, 20 mm MgCl2, 15 mm cel-lobiose, 2 mg of carrier cellulose, and 0.1 /Ci UDP-glucose[glucose-14C(U)] (227 mCi/mmole) or 0.1 /Ci GDP-glucose[glucose-'4C(U)] (203 mCi/mmole). Counts incorporated intolipid soluble and base stable fractions were assayed by washingthe centrifuged pellet three times with hot water, extractingthe lipid soluble fraction twice with 1 ml of chloroform-methanol (2: 1), and removing hot base soluble polysaccharideswith 1 N NaOH, followed by two further water washes. Theremaining base stable residue was collected on a glass-fibrefilter. Filters and chloroform-methanol fractions were driedand counted in toluene based scintillation cocktail. Basesolubilized radioactivity was neutralized with 2N HCI andcounted in Aquasol (New England Nuclear Co.).

Electron Microscopy. Electron microscopy was performedby collecting an appropriate gradient fraction as a centrifugepellet (48,000g, 30 min), prefixing for 15 min with 2%glutaraldehyde in 0.1 M potassium phosphate, pH 7.4, and post-fixation with 1% OsO in 0.1 M potassium phosphate, pH 7.4,for 1 hr. Dehydration was through an ethanol series followedby an ethanol-propylene oxide series. The membrane pelletswere infiltrated overnight with propylene oxide-Epon (1:1)in a vacuum desiccator then embedded in Epon which wascured for 48 hr at 60 C in a vacuum oven. Sections were cutwith a diamond knife and stained with uranyl acetate andlead citrate, followed by ob-servation with a Phillips 200electron microscope.

RESULTS

Partial Purification of Membranous Organeiles on SucroseGradients. The partial purification of the organelles involvedin the secretion of cell wall glycoproteins was performed byusing sequential isopycnic and rate zonal sucrose gradients.When carrot disks are incubated with "4C-proline for 20 min,the newly synthesized cellular proteins become radioactivelylabeled (3). The conversion of "4C-proline to "4C-hydroxyprolineis a post-translational modification mediated by a cytoplasmicpeptidyl proline hydroxylase enzyme (20). In the cytoplasmicproteins, only about 5% of the proline residues are hydroxyl-ated, but the cell wall precursor proteins are extensivelyhydroxylated, and up to 75 or 80% of the proline residuesbecome hydroxyproline residues (3). The percentage of theprotein-bound radioactivity in hydroxyproline residues cantherefore be used as a measure of purification of the cell wallprecursors (2) or the organelles containing these precursors.Homogenates of carrot disks, pulse labeled for 20 min with

"4C-proline, were layered on linear 20 to 50% sucrose gradientsand centrifuged to equilibrium. The resulting UV absorptionprofile (280 nm) and the position of the protein-bound "C-hydroxyproline are shown in Figure 1. The UV absorptionprofile shows a sharp peak at 1.21 g/cm3. These organelles wereidentified as mitochondria by electron microscopy. The largeshoulder at the top of the gradient contained not only thesoluble proteins but also all the orange-colored chromoplastscharacteristic of carrot roots. The newly synthesized cell wallproteins (protein-bound "C-hydroxyproline) were contained ina diffuse band of organelles with an average density of 1.15g/cm3. In this region of the gradient, 35% of the incorporatedradioactivity was in hydroxyproline while 65% was in proline.These figures suggest that only a partial purification of thesecretory organelles had been achieved and that other newlysynthesized proteins were present. An electron micrograph ofthe membranes collected from the "C-hydroxyproline labeledfractions of the gradient is shown in Figure 2. Numerous

Plant Physiol. Vol. 55, 1975 537



GARDINER AN

L

0

'Ox

FIG. 1. Aged carrot tissue disks were labeled for 20 min with 1

ACi of proline-j4C, rinsed, and homogenized in the homogenizationmedium containing 0.5% glutaraldehyde to stabilize dictyosomes.Cytoplasmic membranes were centrifuged through a 20 to 50%sucrose gradient to isopycnic equilibrium and gradients were ana-

lyzed for UV absorbing particles ( ), gradient density (0), andhydroxyproline-'4C in macromolecules (A). The hydroxyproline-rich membranes contain 35% of their total radioactivity in hydroxy-proline, have an average density of 1.15 g/cm3, and are separatedfrom the more dense mitochondria (identified from electron micro-graphs) and the less dense orange band of plastids which float at thegradient-homogenate interface.

;. ...

: O

f:4z.s.: 't .:

A. \

.. X'*: :: : ......... ......8,. , r

*t'... ..A 4 v ^ . : < ..x. ox .,:: K ,4t

^.g _. :'. .. :. +

;

a .. ... e.0 . ... . .......... ....# a > WP " * ' *

K ^ e .. ..

FIG. 2. Electron micrograph of membranous organelles con-

tained in the hydroxyproline-rich isopycnic fraction. Dictyosomes,rough endoplasmic reticulum, and smooth vesicles are evident.

ID CHRISPEELS Plant Physiol. Vol. 55, 1975

membranous structures are detectable, including dictyosomes,rough-endoplasmic reticulum, and smooth membranousvesicles of different sizes.The labeling kinetics of the cytoplasmic protein-bound "4C-

hydroxyproline suggest that most of it is present in cytoplasmiccell wall precursors (3). To show that the "C-hydroxyprolinepresent in these membranous organelles is also in cell wallprecursors, we performed a pulse-chase experiment in whichthe tissue was labeled for 20 min and then chased in 1 mmproline for periods up to 30 min. This experiment showed (Fig.3) that the '4C-hydroxyproline in these organelles is transient,with a half-life of about 10 min. Similar values for cell wallglycoprotein secretion have been obtained by us (3) and byothers (6, 17).

Further purification of the organelles containing the newlysynthesized cell wall proteins was accomplished using ratezonal sucrose gradient centrifugation of the appropriate frac-tions from the isopycnic gradients. The organelles present inthe diffuse UV-absorbing band of the isopycnic gradients werecollected by centrifugation onto a 50% sucrose cushion. Aftera suitable dilution, they were transferred to the top of a linear20 to 35% sucrose gradient and centrifuged at 15,000 rpmfor 20 min (rate-zonal separation). The UV-absorbance profileand the location of the protein-bound '4C-hydroxyproline isshown in Figure 4. Most of the "4C-hydroxyproline is associatedwith organelles that sediment about halfway through thegradient. About 69% of the radioactivity associated with thispeak is in hydroxyproline. This represents a considerableincrease over the previous value (35% in the fractions fromthe isopycnic gradient), suggesting that organelles containingother newly synthesized proline-containing proteins have beenremoved. The figure now approaches the level normally foundin cell walls at which 75 to 80% of the radioactivity is inhydroxyproline. Figure 5 is an electron micrograph of themembranous organelles present in the middle peak of therate-zonal gradient. Most apparent is a considerable enrich-

Chase Time (min)FIG. 3. Pulse-chase experiment in which aged tissue disks were

labeled for 20 min with 1 AtCi of proline-'4C followed by a chaseperiod in 1 mM proline. Tissue disks were homogenized and cyto-plasmic fractions applied to isopycnic gradients. Following centrif-ugation the amount of hydroxyproline-"C in hydroxyproline-richfractions was determined. Radioactivity continues to be incorpo-rated into membrane protein (A) for 2 to 3 min after the chase butthen decreases with a half-life of about 10 min, whereas that incytosol proteins (0) remains low and only decreases slightly.

538

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ment in structurally intact dictyosomes although numerousother membranous structures are also present in this fraction.

lo0

'0x

EQa-

I

81

61

4

B TFIG. 4. Profile of UV absorbing particles ( ) and membra-

nous hydroxyproline-14C (A) on a rate zonal gradient after pre-liminary purification on an isopycnic gradient. Radioactive hydroxy-proline moves coincident with the middle UV absorbing peak andrepresents 69% of the total radioactivity of that band of mem-branes.

Table I. Incorporation of'4C-Sugars from Various Sugar NucleotidePrecursors into Products Soluble in Chloroform-Methaanol

(2:1) or Hot 1 N NaOH or Iinsoluble in Hot BaseThe total membrane pellet of 20-g aged disks was resuspended

in 2 ml of homogenization medium and 100 ,ul were used' in thesubsequent assays. Each reaction mixture (200 pl total volume)contained 100 mm tris, pH 8, 1 mM Na2EDTA, 1 mm- dithiothreitol,20 mM MgCl2, 15 mM cellobiose, 2 mg of carrier cellulose, and su-gar nucleotide: 2 AM UDP-glucose [glucose-'4C(U)] (227 mCi/mmole), 2.5 AM GDP-glucose [glucose-_4C(U)] (203 mCi/mmole),3 JM GDP-mannose [mannose-'4C(U)] (166 mCi/mmole), 2.5 AMUDP-arabinose [arabinose-_4C(U)] (183 mCi/mmole), or 2 AMUDP-galactose [galactose-14C(U)] (254 mCi/mmole). All reac-tions were performed at 35 C for 15 min and were stopped by heat-ing in a boiling water bath for 5 min before washing and extractingthe various fractions as indicated in "Materials and Methods."The data are expressed as the total cpm incorporated into thevarious extractable products and the percentage of total countsincorporated into each fraction.

Chloroform: Base Soluble Base InsolubleMethanol

cpm cprn %5,1 cpnI %UDP-glucose 22,857 92.4 1,010 4.1 862 3.5GDP-glucose 207 10.6 403 20.6 1,346 68.8GDP-mannose 695 7.4 1,437 15.4 7,202 77.2UDP-arabinose 90 6.1 785 53.2 601 40.7UDP-galactose 7,652 88.2 I 6.7 7.1 403 4.6

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ici

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4

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'. !

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B TFIG. 6. Profile of marker enzymes on an isopycnic sucrose gra-

dient: IDPase (0), Cyt-c reductase (0), UDP-arabinose arabinosyltransferase (0), and UV absorbance ( ). IDPase and arabinosyltransferase coincide with the diffuse hydroxyproline-rich band ofmembranes whereas Cyt reductase is intermediate between thehypro-rich membranes and the peak identified as mitochondria.

.4'r

..,

i

# ^.,

..

FIG. 5. Electron micrograph offrom the middle hydroxyproline-riclafter previous partial purification bymembranes are intact dictyosomesdictyosomes by vesiculation.

U.1P

m Location of Marker Enzymes on the Sucrose Gradients.,',,,,,"Appropriate marker enzymes were used to locate the various

membranous organelles on the two types of sucrose gradientsand to further identify the organelles involved in glycoproteinsecretion. Inosine diphosphatase was used as a marker fordictyosomes, Cyt c reductase as a marker for the endoplasmic

h band of a rate zonal gradient reticulum, and UDP-glucose glucosyl transferase, whlch trans-isopycnic sedimentation. Most fers glucose to lipid-soluble products, as a plasma membraneor appear to be derived from marker. UDP-arabinose arabinosyl transferase, an enzyme

known to be involved in the glycosylation of peptidyl-hydroxy-

Plant Physiol. Vol. 55, 1975 539

I



GARDINER AND CHRISPEELS

-1O

E E

fin _

LU_ 0

CuLn1-,

L.u,

lox

EC.

Qu

IL

L-

-U

_lCVC

rDX

) F

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FIG. 7. Profile of marker enzymes on a rate zonal gradient:IDPase (0), Cyt c reductase (0), UDP-arabinose arabinosyl trans-ferase (0), UDP-glucose glucosyl transferase (U), and A ( ).

IDPase and UDP-arabinose arabinosyl transferase coincide withthe intermediate band found to be rich in hydroxyproline. Cyt c

reductase and UDP-glucose glucosyl transferase are found to be as-

sociated with membranes that remain near the top of the gradient.

proline, was used as a marker for the site of glycosylation ofthe hydroxyproline-rich peptide. Preliminary experiments were

done with total membrane fractions to check the transfer ofsugars from sugar nucleotides to endogenous acceptors. Wefound (Table I) that most of the sugar from UDP-Glucoseand UDP-galactose was transferred to chloroform-methanol(2:1) -extractable products (possible glycolipids), whereas thesugars from GDP-mannose, UDP-arabinose and GDP-glucosewere transferred largely to nonlipid molecules.The position of the marker enzymes in the isopycnic

gradients is shown in Figure 6. The organelles containing thenewly synthesized cell wall glycoprotein banded together withthe marker for dictyosomes and with the arabinosyl transferase.The marker for the endoplasmic reticulum occurred in thesame general area of the gradient but had a somewhat greatermean density. The plasma membrane was also found in thisregion of the gradient (data not shown). These data are con-

sistent with the presence of rough endoplasmic reticulum,smooth membranes, and dictyosomes in the electron micro-graph shown in Figure 2. Further purification on rate zonalgradients showed (Fig. 7) that the marker enzymes for theendoplasmic reticulum and the plasma membrane can beclearly separated from the protein-bound "4C-hydroxyprolinewhich remained associated with the IDPase and the arabinosyltransferase.

DISCUSSION

It has long been known that the Golgi apparatus plays an

important role in the biogenesis of the plant cell wall (for a

review see Mollenhauer and Morre [14]), although the precisefunction of this organelle is only now being elucidated,primarily as a result of improved cell fractionation techniques.Recent evidence suggests that the Golgi apparatus is involvedin the biosynthesis and the secretion of the hemicellulosiccomponent of the cell wall (1, 18) and of other extracellularpolysaccharides (9). In animal cells, the Golgi apparatus func-tions in the secretion and terminal glycosylation of extracellu-

lar proteins and mucopolysaccharides (10, 16, 21, 24). Theobservation that the glycosylation of cell wall proteins (11) andthe secretion of the completed glycoproteins (3, 6) is mediatedby cytoplasmic membranous organelles led us to investigatethe possibility that the Golgi apparatus also functions in theprocessing and secretion of extracellular glycoproteins in plantcells. Our results suggest that this is indeed the case.We observed a parallel enrichment in structurally intact

dictyosomes and newly synthesized cell wall protein whencytoplasmic organelles were first fractionated on isopycnicgradients and then further purified on rate-zonal gradients. Inboth types of gradients the newly synthesized cell wall proteinbanded with IDPase, a Golgi apparatus marker enzyme (7, 18),and with UDP-arabinose arabinosyl transferase, an enzymeinvolved in the glycosylation of the polypeptide moieties ofthe glycoprotein (11). The average buoyant density of theorganelles involved in the cell wall glycoprotein secretion wasfound to be 1.15 g/ cm3, a figure which is consistent with thedensity of dictyosomes from pea stems (18). Such evidencesuggests that dictyosomes are involved in both glycosylationof the hydroxyproline-rich protein and the subsequent secre-tion of the glycoprotein. Our results show that the dictyosome-rich fractions containing the hydroxyproline-rich glycoproteincan be separated from smooth membrane fractions containingendoplasmic reticulum and plasma membrane-based onmarker enzyme identifications. Dashek (6) was unable todistinguish between the different types of smooth membranesand vesicles. Roberts and Northcote (19) interpret their resultsto show that the hydroxyproline-rich protein enters the cellwall by intussusception from smooth membranes other thanGolgi vesicles. However, they are not able to differentiatebetween grains derived from proline or hydroxyproline andhave used as a basis for this conclusion a micrograph of aforming cell plate. They also conclude that relatively littlehydroxyproline-rich protein is being deposited in walls ofdividing cells and newly formed cell wall. The discrepancybetween their results and ours may be attributable to the factthat their electron micrographs containing dictyosome vesiclesare of newly forming cell well, in which little hydroxyprolineis deposited, and in which one would not expect to findhydroxyproline-rich proteins. Our results further suggest theimportant role of the Golgi apparatus in the biosynthesis andsecretion of extracellular macromolecules, whether they beglycoproteins of animal or plant cells, mucopolysaccharides ofanimal cells, or hemicellulosic polysaccharides of plant cells.

LITERATURE CITED

1. BOWLES, D. J. AND D. H. NORTHCOTE. 1972. The sites of synthesis andtransport of extracellular polysaccharides in the root tissues of inaize.Biochem. J. 130: 1133-1145.

2. BRYSK, MI. M. AND M. J. CHRISPEELS. 1972. Isolation and partial charac-terization of a hydroxyproline-rich cell wall glycoprotein and its cyto-plasmic precursor. Biochim. Biophys. Acta 257: 421-432.

3. CHRISPEELS, M. J. 1969. Synthesis and secretion of hydroxyproline contain-ing macromolecules in carrots. I. Kinetic analysis. Plant Physiol. 44: 1187-1193.

4. CHRISPEELS, M. J., D. SADAVA, AND Y. P. CHO. 1974. Enhancemiient ofextensin biosynthesis in aging disks of carrot storage tissue. J. Exp. Bot.In Press.

5. DALLNER, G., P. SIEKEvirz, AND G. E. PALADE. 1966. Biogenesis of endo-plasmic reticulum membranes. II. Synthesis of constitutive microsomalenzymes in developing rat hepatocyte. J. Cell Biol. 30: 97-117.

6. DASHEK, W. V. 1970. Synthesis and transport of hydroxyproline-ricli comi1-ponents in suspension cultures of sycamore-maple cells. Planit Physiol.46: 831-838.

7. DAUWALDER, M., W. G. WHALEY, AXD J. E. KEPHART. 1969. Pho-Iliatasesand differentiation of the Golgi apparatus. J. Cell Sci. 4: 455-497.

8. GARDINER, M. G. AND M1. J. CHRISPEELS. 1973. Involveement of thie Golgiapparatus in the synthesis and secretion of hydroxyproline-richl cell wallglycoprotein. Plant Phvsiol. 51: S-60.

Plant Physiol. Vol. 55, 1975




9. HARRIS, P. J. AND D. H. NORTHCOTE. 1971. Polysaccharide formation inplant Golgi bodies. Biochim. Biophys. Acta 237: 56-64.

10. JAMIESON, J. D. AND G. E. PALADE. 1966. Role of the Golgi apparatus in theintracellular transport of secretory proteins. Proc. Nat. Acad. Sci. U.S.A.55: 424-431.

11. KARR, A. L. 1972. Isolation of an enzyme system which will catalyze theglycosylation of extensin. Plant Physiol. 50: 275-282.

12. LAMPORT, D. T. A. 1965. The protein component of primary cell walls. Adv.Bot. Res. 2: 151-218.

13. LAMPORT, D. T. A. 1969. The isolation and partial characterization of hydroxy-proline-rich glycopeptides obtained by enzymatic degradation of primarycell walls. Biochemistry 8: 1155-1163.

14. NIOLLENHAUER, H. H. AND D. J. MORRE. 1966. Golgi apparatus and plantsecretion. Annu. Rev. Plant Physiol. 17: 27-46.

15. MORRE, D. J., H. H. MOLLEXNHAER, AND J. E. CHAMBERS. 1965. Glutaralde-hyde stabilization as an aid to Golgi apparatus isolation. Exp. Cell Res.38: 672-675.

16. NEUTRA, 'M. AND C. P. LEBLOND. 1966. Radioautographic comparison of theuptake of galactose-3H and glucose-3H in the Golgi region of various cellssecreting glycoproteins or mucopolysaccharides. J. Cell Biol. 30: 137-150.

17. OLSON, A. C. 1964. Proteins and plant cell walls. Proline to hydroxyproline intobacco suspension cultures. Plant Physiol. 39: 543-550.

541

18. RAY, P. 'M., T. L. SHININGER, AND M. M. RAY. 1969. Isolation of 8-glucansynthetase particles from plant cells and identification with Golgi mem-

branes. Proc. Nat. Acad. Sci. U.S.A. 64: 605-612.19. ROBERTS, K. AND D. H. NORTHCOTE. 1972. Hydroxyproline: observations on

its chemical and autoradiographical localization in plant cell wall protein.Planta 107: 43-51.

20. SADAVA, D. AND M. J. CHIRISPEELS. 1971. Hydroxyproline biosynthesis inplant cells. Peptidyl proline hydroxylase from carrot cells. Biochim. Biophys.Acta 227: 278-287.

21. SCHACHTER, H., I. JABBAL, R. L. HUDGIN, L. PINTERIC, E. J. McGuIRE, AND

S. ROSEMAN. 1970. Intracellular localization of liver sugar nucleotide glyco-protein glycosyltransferases in a Golgi-rich fraction. J. Biol. Chem. 245:1090-1100.

22. TALSSKY, H. H. AND E. SHORR. 1953. A microcolorimetric method for thedetermination of inorganic phosphorus. J. Biol. Chem. 202: 675-685.

23. VAN DER WOtDE, W. J. 1973. Significance of the specific staining of plantplasma membranes by treatment with chromic acid-phosphotungstic acid.Plant Physiol. 51: S82.

24. WAGNER, R. R. AND M. A. CYNKIN. 1971. Glycoprotein biosynthesis. In-corporation of glycosyl groups into endogenous acceptors in a Golgiapparatus-rich fraction of liver. J. Biol. Chem. 246: 143-151.

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Involvement Golgi Apparatus Synthesis Secretion of ... · Plant Physiol. (1975) 55, 536-541 Involvement ofthe Golgi Apparatus in the Synthesis andSecretion ofHydroxyproline-rich Cell